Monoclonal origin of vulvar intraepithelial neoplasia and some vulvar hyperplasias.

Squamous neoplasms of the female genital tract, including vulvar intraepithelial neoplasia, presumably are derived from a single cell. This study addressed this hypothesis and determined the clonal status of other squamous epithelial alterations associated with vulvar carcinoma, including hyperplasia and lichen sclerosis. X chromosome inactivation patterns of 22 epithelial lesions and matched normal epithelium were determined using a polymerase chain reaction (PCR)-based assay targeting the X-linked human androgen receptor gene (HUMARA). Clonality was inferred by comparing matched lesional and control tissues as follows: 1) monoclonal, if intensity of either PCR product was skewed relative to normal reference epithelium (control), 2) polyclonal, if both lesional and control were unskewed, and 3) unknown, if both lesion and control tissues were skewed toward the same allele. Two cases were excluded because of noninformative homozygous HUMARA alleles. Of 8 vulvar intraepithelial neoplasias analyzed, 7 were scored monoclonal and 1 polyclonal. Of 12 hyperplasias, 6 were monoclonal, including one with lichen sclerosis, 2 were polyclonal, and in 4, the clonal status could not be determined. The PCR-based clonal assay supports a monoclonal derivation for vulvar intraepithelial neoplasia and, in some cases, vulvar hyperplasia, and lichen sclerosis. The finding of monoclonal hyperplasia and lichen sclerosis suggests that clonal expansion may evolve before the development of morphological atypia in these epithelia.     

Development of a PCR-SSOP approach capable of defining the natural killer cell inhibitory receptor (KIR) gene sequence repertoires

A molecular typing method based on polymerase chain reaction (PCR) amplification of three different target domains (immunoglobulin domains 1 and 3, and the transmembrane-cytoplasmic domain), followed by hybridisation with 26 digoxigenin-labelled sequence-specific oligonucleotide probes (SSOP) has been established for the polymorphic killer inhibitory receptor (KIR) genes. In addition to identifying the 12 KIR subfamilies, our PCR-SSOP typing approach could also distinguish the putative alleles, NKB1 and NKAT3, that comprise the KIR3DL1 subfamily. Ninety unrelated blood donors and 13 families (52 individuals), including both parents, were subjected to our KIR PCR-SSOP typing approach. All 12 KIR subfamilies, including a 2DS5 variant sequence, were present in the 90 individuals and displayed varied phenotype frequencies: 2DL1 (0.96), 2DL2 (0.31), 2DL3 (0.95), 2DS1 (0.56) 2DS2 (0.51), 2DS3 (0.27), 2DS4 (0.96), 2DS5v (0.35), 3DS1 (0.47), 3DL1 (0.96), 3DL2 (1.0) and 2DL4 (1.0). A total of 23 different KIR phenotypes were defined in this study, and 10 of these were only found on one occasion in one individual, indicating considerable diversity in the KIR phenotype profiles within the Irish population. Most individuals (93%) possessed the complement of inhibitory KIR specificities for the three well-defined HLA-B and -C ligands. An unusual probe pattern for 3DS1 was observed in 3 individuals indicating a variant 3DS1 gene sequence with changes at nucleotide positions 1185-1186, within the cytoplasmic domain. Sequencing analysis revealed a new single nucleotide polymorphism in exon 3 of 3DL1 NKB1(195, G-A) and a 22-bp deletion polymorphism in exon 5 of 2DS4 (nucleotides 777-798 deleted). A number of strong KIR associations were observed, namely 2DL1 with 2DL3, 2DS4 with 3DL1, 2DL2 with 2DS1/2DS2/2DS3, 2DS1 with 2DS3/2DS5v/3DS1, 2DS2 with 2DS3 and 2DS5v with 3DS1. Analysis of the KIR segregation observed in the 13 families confirmed these strong associations and permitted the definition of a number of partial KIR haplotypes, e.g. 2DL2-2DS1-2DS2-2DS3-3DL1. The segregation analysis concluded that at least 3 distinct gene loci encode 2DL1-4 and at least 4 gene loci encode the non-inhibitory KIR2DS1-2DS5. In the case of 3DL1-2 and 3DS1, our data suggests 3 gene loci, one for each subfamily.     

Allelic loss in human papillomavirus-positive and -negative vulvar squamous cell carcinomas

Vulvar squamous cell carcinoma (VSCC) is a biologically and morphologically diverse disease, consisting of human papillomavirus (HPV)-positive and -negative tumors that differ in their morphological phenotypes and associated vulvar mucosal disorders. This study analyzed the frequencies of allelic loss (loss of heterozygosity (LOH)) in HPV-positive and -negative VSCCs to identify potential targets for the study of preinvasive diseases, to determine whether HPV status influenced patterns of LOH, and to determine whether these patterns differed from HPV-positive tumors of another genital site, cervical squamous cell carcinomas (CSCC). DNA extracted from microdissected archival sections of two index tumors, one each HPV negative and positive, was analyzed for LOH at 65 chromosomal loci. Loci scoring positive with either sample were included in an analysis of 14 additional cases that were also typed for HPV. Frequencies of LOH at loci were computed in a panel of HPV-positive and -negative VSCCs. Twenty-nine loci demonstrated LOH on the initial screen and were used to screen the remaining 14 tumors. High frequencies of LOH were identified, some of which were similar to a prior karyotypic study (3p, 5q, 8p, 10q, 15q, 18q, and 22q) and others of which had not previously been described in VSCC (1q, 2q, 8q, 10p, 11p, 11q, 17p, and 21q). With the exception of 5q and 10p, there were no significant associations between frequency of LOH and HPV status in VSCC. LOH at 3p and 11q were frequent in both VSCC and CSCC; however, allelic losses at several sites, including 5q, 8q, 17p, 21q, and 22q, were much more common in VSCC. VSCCs exhibit a broad range of allelic losses irrespective of HPV status, with high frequencies of LOH on certain chromosomal arms. This suggests that despite their differences in pathogenesis, both HPV-positive and -negative VSCCs share similarities in type and range of genetic losses during their evolution. Whether the different frequencies of LOH observed between VSCC and CSCC are real or reflect differences in stage and/or tumor size remains to be determined by further comparisons. The role of these altered genetic loci in the genesis of preinvasive vulvar mucosal lesions merits additional study.     

A comparison of biotin and isotope-labeled ribonucleic acid probes for in situ detection of HPV-16 ribonucleic acid in genital precancers

Genital precancers were analyzed by in situ hybridization for the presence of HPV RNA using single-stranded RNA probes. To compare the sensitivity of biotin- and 35S-labeled probes, serial sections from three biopsies containing variable amounts of HPV 16 RNA were analyzed with probes containing each label. The probe template was a cloned fragment of the HPV 16 genome spanning portions of the E2, E5 and L2 orf's. When a focus contained a strong hybridization signal with 35S-labeled probes (i.e., target/background ratio greater than 40) serial sections of the same area usually demonstrated a strongly positive signal with biotin-labeled probes. When the signal with 35S in a given focus was weak (target/background ratio less than 20), serial sections contained either a very weak or no detectable signal with the biotinylated probe. From this study it appears than 35S-labeled RNA probes exceed the sensitivity of biotin-labeled probes by nearly an order of magnitude with short exposures (4 days), and with longer exposures (1 to 2 weeks), approach the sensitivity of that reported for tritiated probes and exposures of 1 month or longer.     

Expression of candidate tumor markers in ovarian carcinoma and benign ovary: evidence for a link between epithelial phenotype and neoplasia

EpCAM, epithelial membrane antigen (EMA)-mucin 1 (MUC1), mesothelin, and CD9 have been reported to be overexpressed at the RNA level in ovarian carcinomas. By using immunohistochemistry, we profiled the protein expression of these gene products in ovarian carcinoma tissues and compared them with benign ovarian surface epithelium (OSE) and cortical inclusion cysts (CICs). Immunoreactivity for EMA and calretinin were used to define epithelial and mesothelial differentiation in nontumor tissues, respectively. Papillary serous (n = 16) and endometrioid (n = 10) tumors were immunopositive for EMA/MUC1 (100%), mesothelin (75% and 30%, respectively), CD9 (88% and 90%, respectively), and EpCAM (100%). All ovarian carcinomas and carcinoma cell lines tested were negative for calretinin. In nonneoplastic ovary, both OSE and CICs ranged from flat-to-cuboidal to stratified and ciliated in appearance. OSE with a cuboidal morphology had a similar immunoreactivity as omental peritoneum, expressing calretinin, mesothelin, and CD9. In contrast, CICs with stratified and ciliated epithelium show expression patterns similar to those in fallopian tubes. They frequently expressed EMA, EpCAM, mesothelin, and CD9. This immunophenotype is preserved in ovarian carcinomas, suggesting that Müllerian metaplasia signals the acquisition of these markers and that their expression is maintained in ovarian carcinomas that originate from this epithelium.     

Early vulvar squamous neoplasia: advances in classification, diagnosis, and differential diagnosis

The recognition and classification of preinvasive vulvar neoplasia are complicated by the facts that (a) their respective carcinomas have a diverse (human papillomavirus [HPV]- and non-HPV-related) pathogenesis; (b) not all vulvar squamous carcinomas are associated with precursors with strictly defined morphologic features; (c) many carcinomas have epithelial changes that are abnormal but lack sufficient nuclear atypia to warrant classification as an intraepithelial neoplasm; and (d) even lesions associated with a common etiologic agent (HPV) present a diverse morphologic spectrum. In this review, five categories of early vulvar neoplasia are defined, based on the available literature, into (a) low-grade lesions with minimal cancer risk, (b) high-grade lesions associated with HPV, (c) high-grade lesions associated with other etiologies, (d) squamous atypias defined by abnormalities in differentiation rather than abnormalities in nuclear morphology, and (d) early carcinomas that do not exhibit conspicuous stromal invasion. The first three groups are arranged into low- and high-grade intraepithelial lesions, the fourth into intraepithelial atypias that bear careful follow-up and attention to the co-existing squamous mucosa, and the fifth into a category that, depending on the degree of cell differentiation, may warrant local excision or lymph node dissection. Recognition of these five categories is germane to proper management of women with squamous lesions of the vulva.     

Desensitization of the adipocyte A(1) adenosine receptor during untreated experimental diabetes mellitus

We examined the changes that occur in the adenosine receptor system during diabetes mellitus. Experimental diabetes mellitus was induced in male Lewis rats with streptozocin (65 mg/kg), and A₁ adenosine receptor binding was characterized with [¹²⁵I]N ⁶-2-(4-aminophenyl) ethyladenosine. In adipocytes, high-affinity A₁ adenosine receptor binding decreased from 1466±228 of protein to 312±123 fmol/mg of protein (p<0.01) following 14 d of untreated diabetes mellitus. Neither the dissociation constant (K _d=1.3±0.2 nM) nor the basal level of adenylate cyclase activity (2.8±1.1 pmol cAMP/mg of protein/min) was altered by diabetes mellitus. The dose-response curve for the inhibition of adenylate cyclase byN ⁶-R-phenylisopropyladenosine (R-PIA), however, did show a rightward shift, indicating that diabetic adipocyte membranes were less sensitive to the effects of adenosine than nondiabetic adipocyte membranes. In contrast, the A₁ adenosine receptor-binding characteristics and adenylate cyclase dose-response curve for cerebral cortical tissue were unchanged by diabetes. These findings suggest that diabetes has tissue-specific effects on the A₁ adenosine receptor system. Furthermore, the decreased sensitivity to adenosine potentially worsens the hyperlipidemia associated with diabetes mellitus. Such alterations in the adenosine receptor system may play a previously undescribed role in the pathophysiology of diabetes mellitus and may help explain why some organs are severely affected by diabetes, but others are relatively spared. Understanding these alterations in adenosine receptor function may lead tonovel therapies of this common metabolic disease.     

Empirical examination of current depression categories in a population-based study: symptoms, course, and risk factors

OBJECTIVE: Research studies on the validity of current diagnostic and subthreshold categories of depression that use a population-based follow-up design are rare. The authors examined the validity and utility of four current depression categories by examining subject transition between categories and the symptoms, course, and risk factors of each. METHOD: A general population sample of 1,920 adults from the Baltimore Epidemiologic Catchment Area 13-year follow-up study were examined. Data on diagnoses, symptoms, course, and risk factors were collected by using the National Institute of Mental Health Diagnostic Interview Schedule, the Life Chart Interview, and an office visit. Polychotomous regression was used to examine the heterogeneity of four diagnostic categories: major depressive disorder, depressive syndrome, dysthymia, and a comorbid depression condition (major depressive disorder and dysthymia). RESULTS: Transitions between the four depression categories occurred over the 13 years. Symptom profiles for the four categories were parallel but differed in severity. Course characteristics among the four categories slightly differed. Risk factor profiles showed significant differences. Family history was associated with both depressive syndrome and major depressive disorder. Stressful life events were most strongly associated with depressive syndrome. Female gender was most strongly associated with the comorbid depression category. CONCLUSIONS: The evidence suggests that except for dysthymia, the depression categories are genetically homogeneous and environmentally heterogeneous. Stress is associated with mild depression, and gender is associated with severe depression. The apparent familial transmission of the subthreshold entity, depressive syndrome, needs further investigation.